SBB Last Chance Review 2017 - South Central … Last Chance Review 2017. Carter BloodCare, ... Phyllis Kirchner, MSTM, MT (ASCP) SH, SBBCM . Sue Johnson, MSTM, MT (ASCP) ... maximum

SBB Last Chance Review

Review for the SBB/BB Exam

March 25-26, 2017

Hosted by:

2205 Highway 121,

Bedford, Texas 76021 www.carterbloodcare.org

Presented by:

www.scabb.org www.bcw.edu

© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin

http://www.carterbloodcare.org/

http://www.scabb.org/

http://www.bcw.edu/

SBB Last Chance Review 2017 Carter BloodCare, Bedford, TX

A Big Thank You to Our Faculty

Katrina Billingsley, MT(ASCP)SBB LifeShare Blood Centers, Shreveport, LA

Brenda Barns, MT (ASCP) SBB

Allen College - UnityPoint Health, Waterloo, IA

Cindy Piefer, MT (ASCP) SBB Phyllis Kirchner, MSTM, MT (ASCP) SH, SBBCM

Sue Johnson, MSTM, MT (ASCP) SBB BloodCenter of Wisconsin, Milwaukee, W

Tina Ipe, MD, MPH

Houston Methodist Hospital, Houston, TX

Meredith Reyes, MD St. Luke’s Health, Houston, TX

Lesley Kresie, MD, D (ABHI)

Mike Newhouse, MT(ASCP)SBB Carter BloodCare, Bedford, TX

Heather vonHartitzch, MT(ASCP)SBB Oklahoma Blood Institute, Tulsa, OK

Sandra Nance, MS, MT(ASCP)SBB

Margaret Keller, PhD ARC Philadelphia, Philadelphia, PA

Jessica Drouliard, MLS(ASCP)CMSBBCM

Heartland Blood Centers, Chicago, IL

Continuing Education Credit Up to 16 P.A.C.E. & FL CE credit hours approved

Level of Instructions: Intermediate

SCABB is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E.® Program & FL CE Broker.


SBB Last Chance Review 2017 Carter BloodCare, Bedford, TX

Saturday- March 25, 2017

Start Minutes Topic Presenter

8.00 20 Testing tips: SBB/BB exam Phyllis Kirchner 8.20 30 ABO/Lewis/P1PK/I Sue Johnson 8:50 30 Rh Sue Johnson 9:20 10 Break 9:30 30 Kell, Kidd, Duffy Jessica Drouliard

10:00 30 MNS and other blood groups Cindy Piefer 10:30 40 DAT and Warm Autos Sue Johnson 11:10 30 Lunch 11:40 15 Polyagglutination Katrina Billingsley

11:55 50 Crossmatch and antibody cases Katrina Billingsley

12:45 50 HLA, HPA, HNA, & HSCT Dr. Lesley Kresie 1:35 10 Break 1:45 40 Genetics/population genetics Brenda Barnes 2:25 35 Immunology & complement Brenda Barnes 3:00 40 Adverse effects of transfusion Brenda Barnes 3:40 10 Break

3:50 50 Lab math, lab management, QA and safety Phyllis Kirchner

4:40 10 End Saturday session

4:50 60 Math Boot Camp Bonus (Optional)

Heather vonHartitzch, Sue Johnson, Phyllis Kirchner, Jessica Drouillard

6:30 TBD Networking Dinner (Optional) - TBD

Sunday- March 26, 2017 Start Minutes Topic Presenter

8:00 50 Special techniques Sandra Nance 8:50 30 HDFN & RhIG Mike Newhouse 9:20 30 Blood collection Heather vonHartitzch 9:50 10 Break

10:00 50 Hemostasis and coagulation cases Meredith Reyes, PhD

10:50 50 Blood components and transfusion practice Meredith Reyes, PhD

11:40 30 Lunch 12:10 40 Cell survival & anemias Phyllis Kirchner 12:50 30 Molecular Genotyping Margaret Keller, PhD 1:20 10 Break 1:30 40 TTD testing and re-entry Tina Ipe, MD, MPH 2:10 40 Hemapheresis Tina Ipe, MD, MPH 2:50 10 Closing


Preparing for the ASCP BOC SBB/BB Exam

Phyllis Kirchner, MSTM, MT(ASCP)SH, SBBCM

BloodCenter of Wisconsin

Part 1

ASCP BOC Exam Model Computer Adaptive Test (CAT)

2

BOC at a Glance ASCP Board of Certification (BOC) fee:

$290 SBB: Specialist in Blood Banking $240 BB: Technologist in Blood Banking

Exam Process • Once approved by ASCP, take exam within 3

months • 2½ hours, 100-questions, all multiple-choice • One question presented at a time • Computer adaptive exam (CAT), criterion-

referenced • Passing 400, maximum score 999

3

Exam Content (%) BB SBB

BP Blood products 15-20 15-20

GRPS Blood Group Systems 15-20 15-20

IMMU Immunology 5-10 5-10

LO Laboratory Operations 5-10 15-20

PHYS Physiology/Pathophysiology 5-10 10-15

SER Serological/Molecular testing 20-25 20-25

TRNS Transfusion Practice 15-20 15-20

SBB

***

***

SER the highest – but only 25% at most

Top 5 topics: SER, BP, GRPS, LO, TRNS (LO ↑ for SBB exam)

0 5 10 15 20 25 30

1

2

3

4

5

6

7SBB BB

4

Cognitive Skills Level What is this?? Example:

1 Recall Recall previously learned (memorized) knowledge

What is the most common blood group?

2 Application Use recalled knowledge to

interpret or apply written, numeric or visual data

A patient with sickle cell disease types R0. What antibodies can this patient make?

3 Analysis/

Synthesis/

Evaluation

Apply recalled knowledge to resolve a problem and/or to make a decision

Given discrepant ABO testing results, select the next procedures to resolve the problem

5

Computer-Adaptive Testing (CAT)

• Computer interactive to individual’s ability, and not influenced by other test takers

• The first question is of average difficulty (half of test takers are expected to get right)

• After a few questions, CAT determines your skill level

and will approximate your final score.

CAT tailors questions to match

your ability

Answered correctly: Next question has a slightly higher level of difficulty and

continues until you answer incorrectly

Answered incorrectly: Next question will be slightly easier. This is how it is tailored to

one’s ability level

6


1

Test Tips

CAT Strategy Must answer enough difficult questions correctly to achieve a score above the a passing score (400). Score calculated by:

–Total number of correct answers –Difficulty level of all 100 questions

Strategy: –Do your best to answer each question correctly. This way, you will have a exam that has a high level of difficulty (requiring fewer correct answers to pass) –Guessing (and coming back to it later at review) will cause the computer to give you a test with a lower difficulty and requiring more correct answers to pass 7

Part 2

Application to ASCP BOC

8

Check your eligibility – new for 2017!!! SBB BB

1 Baccalaureate degree…AND successful completion of a CAAHEP-accredited Specialist in Blood Bank Technology program within the last 5 yrs

MT/MLS(ASCP) certification, AND a baccalaureate degree from a regionally accredited college/university

2 MT/MLS(ASCP) or BB(ASCP) certification, AND a baccalaureate degree…AND 3 years of full time acceptable experience in a clinical* laboratory in blood banking… or educator…within the last 10 6 yrs

Baccalaureate degree… with a major in biological science or chemistry … AND 1 year full time acceptable clinical laboratory* experience in blood banking…within the last 10 5 yrs

3 Masters or Doctorate…. degree…AND 3 years of full time acceptable experience in a clinical* laboratory in blood banking… or educator…within the last 10 6 yrs

BS degree…AND completion of BB … under the auspices of a NAACLS accredited MLS Program …within the last 5 yrs

4 Doctorate…AND 2 yrs fellowship in BB…within the last 10 5 yrs

Master’s or doctorate …AND…within the last 10 5 yrs

9

Documents: Transcripts and Experience

Official transcripts* • BS degree only, in a sealed

envelope signed by the college/university

• Academic work completed outside of the U.S. and Canada: must be evaluated by an evaluation agency

Experience

*SBB applicants: Transcript is NOT required if previously certified as a MT/MLS or BB on or after 1/1/2000 Must supply your Certification Number on your application.

Route 1(CAAHEP SBB program) Routes 2, 3, 4

No need to send experience documentation

1. Experience documentation forms 2. Letter of authenticity

10

When to apply

CAAHEP SBB program

• Processing takes 45 days, so plan ahead • Students should apply no earlier than four

to six weeks prior to completing the program.

• SBB program official must sign the ‘record release’

• You must finish all program requirement!

11

Application: CAAHEP SBB Program • Information to complete online application include: • Applicant –Name* as it appears on driver’s license/state ID –Personal email address • SBB program –Name of program (select from drop-down menu) –Program beginning date and ending date –Program director: name, phone # and email address

*If the first and last names do not match the valid ID when the applicant appear at the test center, she/he will not be permitted to take the examination. You will have to reapply and submit a full application fee.

12


2

Test Tips

When to apply Other Routes -Allow time to obtain the following: • Transcripts (and evaluation if applicable) • Experience documentation form • Letter of authenticity

– Experience documentation forms must be completed by your immediate supervisor/laboratory director a

– Attached to a letter of authenticity signed by this individual verifying the authenticity of the form.

– Letter must be printed on original letterhead and state that the experience documentation form was completed by the employer, with date and signature.

– Experience documentation forms will be audited to verify authenticity.

13

Example

14

Serologic Testing • ABO and Rh typing • Antibody detection and

identification • Crossmatching • Direct antiglobulin tests • Test for other blood group

antigens Routine Problem Solving • Transfusion reactions • Immune hemolytic anemias • Indications for transfusion • Hemolytic disease of the fetus

and newborn (HDFN) • Rh immune globulin studies

Quality Control/Quality Assurance • Reagents, equipment Laboratory Operations Donor Collection, Processing and Testing (Proficiency may be demonstrated through performance, observation or simulation) • Donor selection, preparation and

collection • Processing and donor testing • Component preparation for storage

and administration

Clinical Experience

15

Must Apply Online

Online screens are self-explanatory

You can pay by credit card online (ASCP will acknowledge your application within 1 business day) OR

If unable to pay online with a credit card you will get pay-by-mail instructions after completing the online application

You must create an account

if you do not have one

16

Using mail?

Always use regular mail • Do not FAX • Do not use Express or Registered Mail,

FedEx. UPS, ETC

17

ASCP Approval Notification • ASCP will email “Admission Notice” with instructions • Immediately make an appointment (by phone or

online) at Pearson to reserve your test date • You will have 3 months to take the exam

Example of email from ASCP

18


3

Test Tips

Part 3

How to Study

19

Study References

ASCP

• Online Practice Tests • BOC Study Guide • Examination Content Guidelines AABB

• Technical Manual – all chapters plus Methods sections

• Standards 20

The 7 Categories and their Weight BB SBB

BP Blood products 15-20 15-20

GRPS Blood Group Systems 15-20 15-20

IMMU Immunology 5-10 5-10

LO Laboratory Operations 5-10 15-20

PHYS Physiology/Pathophysiology 5-10 10-15

SER Serological/Molecular testing 20-25 20-25

TRNS Transfusion Practice 15-20 15-20

SBB

***

***

SER the highest – but only 25% at most 0 5 10 15 20 25 30

1

2

3

4

5

6

7 SBB

BB

21

Examination Content Guidelines Example: Serologic/molecular testing is 20-25% for SBB exam. What materials are covered?

22

Subsections Examples

Routine tests AABB Standards, compatibility testing, antibody ID, DAT

Reagents AHG, reagent antisera and cells Application of special tests and reagents

Enzymes, adsorption, elution, ELISA, molecular techniques

Leukocytes/platelet testing

Cytotoxicity, platelet testing, granulocyte testing, molecular techniques

Quality assurance Blood samples, reagents, procedures

How to Study: Have a Plan • Gather and organize resources (reading list,

lectures/notes, previous tests, texts, etc) • Identify areas that need additional study • Create a study outline • Develop a comprehensive study schedule. It

may be better to study a short time every day than to a long time infrequently

• Practice answering multiple-choice questions • Allow sufficient time for final review before exam

23

Part 4

Testing Site Taking the Exam After the Exam

24


4

Test Tips

Day of Exam: Bring ID and Letter Day Before Exam

• Pack ID and paperwork

• Know directions to the test site. Do a dry run and find the room

• Relax • Get a good night’s

sleep

Gather these documents:

• ASCP BOC admission letter

• Drivers license (or state ID card) with photo and signature. First/Last name on card must match the registration at Pearson.

• Bring non-programmable calculator

25

Arrival & Checking in at Exam Site • Eat a good breakfast/meal • Arrive at least 30 minutes before test time • At Pearson’s

– Show admission letter and ID – Will take: Signature, photo, palm vein image

• Checklist with rules provided • Not allowed in test room (Place into a locker)

– Personal belonging (including watch, cell phone, large jewelry, etc)

• Another palm vein scan before you are seated in the exam room 26

Testing site: What’s Provided?

• Computer & seating location assigned • Monitoring system – audio and video recorded • 2½ hours, 100-question (90 seconds per

question)

27

Dry Erase Board Antibody Panels

Remember You can bring a non-programmable calculator

Taking the Exam • At computer, verify your name and examination category • Directions will pop up: read carefully • When ready, click START. Time will count down • Questions will appear one at a time • Answer each question best you can. Select A, B, C or D

– You may change response within the question – If unsure of answer, mark it for review – Press the ENTER key or click NEXT. The next

question will then appear • Proctor can be summoned during test

28

Taking the Exam Do your best to select the correct answer, especially the first third of exam • Less difficult questions will require your to answer

more questions correctly to pass • Score is combination of total number of correct

answers and difficulty of correct answers What if you don’t know the answer? • When you guess and enter the wrong answer, CAT will

adjust levels of questions that may be all wrong for your ability level

• Do your best and give the most educated response • Mark the question for review

29

Reviewing/Submitting the Exam Review the exam

– Most people finish the exam in 2 hours – If you have 30 minutes left, review all questions – If less than 30 minutes left, review only marked

questions Changing answers

– Change your answer only if you are sure – When you change an answer, you have 66% of getting it

right. Submit the exam

– A verify screen will appear – click YES – The preliminary PASS or FAIL message will show

30


5

Test Tips

Score Report Score in 4 business days • ASCP will email you to login to get the score report • Report will show scaled score (how many points) • Print the report – this is only document with your

certification number. Otherwise, will have to pay $15 to get the report.

• Individual scores from each category provided to: – SBB program officials – Those who failed exam - provide the areas that need

concentration Certificate • Mailed in 3-6 weeks • Valid for 3 years

31

Did Not Pass? ASCP will send by email • Scaled scores in each subtest • Instructions on how to reapply • May not retake exam within the same three month period Reapplication documents/fee • ASCP keeps your original application and the supporting

documentation for 5 years • Can take 5 times for each eligibility route • Must pay application fee each time Re-apply: takes about 2 weeks for ASCP approval

32

BOC Pass Rate (%)

33

2005

20

06

2007

20

08

2009

20

10

2011

20

12

2013

20

14

2015

20

16

1st time CAAHEP

SBB pass (79%)

BB pass (70%)

SBB total pass

(46%)

86

63

86

77

84

68

84

46

54

62 67

61

50

47

57

Questions for ASCP BOC

ASCP BOC

800-267-2727 [email protected]

34


6

Test Tips

mailto:[email protected]

I, ABO, Hh, LE, P1Pk, Globoside Blood Group Systems

i Collection Sue Johnson, MSTM, MT(ASCP)SBB

Director, Clinical Education BloodCenter of Wisconsin

Milwaukee, WI

Objectives

• Describe the inheritance & molecular basis of ABO, Hh, I, Lea/Leb, P1Pk, Globoside blood group antigens and i collection.

• Recognize the biochemical make-up of ABO, Hh, Ii, Lea/Leb, P1Pk and P.

• Discuss the serological characteristics and clinical significance of antibodies to ABO, Hh, I, Lea/Leb, P1Pk, Globoside blood group antigens.

• List causes for ABO discrepancies & possible steps to resolution.

• Predict the presence of blood group antigens in secretions and on the red cells based on knowledge of genetics.

If you study to remember, you will forget.

If you study to understand,

you will remember!

Unknown

Type II Unbranched Chain

i Antigen

R

ß 1-4

β1,3

β1,4

Biochemistry Basics

α linkage – hydroxyl group attached to no. 1 carbon is below the plane of the ring

OH

β linkage – hydroxyl group attached to no. 1 carbon is above the plane of the ring

OH

Glycolipids – attached to a ceramide Glycoproteins – attached to a serine or threonine, majority of ABO are glycoproteins (Band 3)

1

1

2

2 3

3

4

4

5

5

Type II Unbranched Chain or i Antigen


7

CHO Antigens

Conventional Name

ISBT Symbol

ISBT Number

Antigen Conventional

Name

ISBT Antigen Number

ISBT Full Code Number

I blood group I (27) 27 I 001 027001 or 27.1

Ii collection I (207) 207 i 002 207002

I Blood Group System ISBT Terminology

Genetics of I

• GCNT2*01 (GCNT2 or IGnT or I gene)

• 5 Exons

• 3 alternate forms of exon 1

• 1A, 1B, 1C

• 2 & 3

• 3 isoforms code for transferases affecting different tissues

1A 1B 1C 2 3

Adapted from AABB Technical Manual 18th edition

Genetics of I Isoforms

1A 1B 1C 2 3

1A 2 3 1B 2 3 1C 2 3

IGnTA IGnTB

IGnTC Lens Epithelium RBC

Genetics of i • i adult phenotype without cataracts

– Point mutations in GCNT2 in exon 3

– 6 point mutations

– Autosomal recessive inheritance

– No cataracts

• i adult phenotype with cataracts

– Point mutations or deletion of GCNT2 in exon 2 and 3

1C 2 3

1C 2 3 1C or

Molecular Basis of I

I (GCNT2) Chromosome 6

6-β-N-acetylglucosaminyltransferase

(β-6GlcNAc-transferase), or

β-1,6-acetylglucosaminyltransferase

“Branching Enzyme”

i Antigen

R

ß 1-4 β1,3

β1,4

Straight Type II Chain


8

CHO Antigens

I Antigen

R

ß 1-4 β1,3

β1,4

β-1,6-acetylglucosaminyltransferase

Branched Type II Chain

Type II Branched Chain or I Antigen

Developmental Differences of the I & i Antigen and Their Association with ABH

Newborn

H

I

A B

i

Adult

Courtesy of A. Rossier

Characteristic I i

Gene IGnT or GCNT2

(ISBT terminology) Unknown

Oligosaccharide chain

Branched Unbranched

Antigen sites 32,000 to 500,000 20,000 to 70,000

Location in RBC membrane

Glycoproteins, glycolipids, Band 3

Glycoproteins, glycolipids, Band 3

Body Fluids Saliva, milk, amniotic fluid,

urine, ovarian cyst Saliva, milk, amniotic

fluid, urine, ovarian cyst

Other blood cells Lymphocytes, monocytes,

granulocytes, platelets Lymphocytes, monocytes,

granulocytes, platelets

Adult Strong (complete conversion

around 2 years old) Weak

Cord Cell Weak Strong

iadult Trace Strong

ANTIGENS DEPENDENT ON DETERMINANTS OF MORE THAN ONE BLOOD GROUP SYSTEM

IA

IB

IH

iH

IP1

ITP1

iP1

IP

ILebH

IBH

IAB

iHLeb

Characteristic Autoanti-I Alloanti-I Autoanti-i

Ig Class IgM IgM IgM

Temp. of Reactivity

RT to 4C – Benign 30 – 37C - Pathologic

RT to 4C Rarely 37C

RT to 4C – Benign 30 – 37C - Pathologic

Bind Complement

Rarely Rarely Rarely

Hemolysis in vitro

No No No

Enzyme Treated RBCs

HTR No None to Rare No

HDFN No No No

Adult Strong + Strong + Weak +

Cord Cell Neg to Weak + Neg to Weak + Strong +

iadult Trace Negative Strong +

Antibody Characteristics


9

CHO Antigens

Identification of Cold Autoantibodies

RBCs Tested

Antibody Specificity

A1

A2

B

O

O cord

Auto

Comments

Anti-I 4+ 4+ 4+ 4+ 0-2+ 4+

Anti-IH 0-2+ 3-4+ 0-2+ 4+ 0-2+ 0-2+ Found in A1B, A1 & B

Anti-i 0-2+ 0-2+ 0-2+ 0-2+ 4+ 0-2+

Techniques

• Wash cells with warm saline

• Prewarm

• Cold panel (Adult cells and cord cells incubated at different temperatures (RT, 150C and 40C)

• Autoadsorption

• Rabbit Erythrocyte Stroma

– Adsorb cold autoantibodies

– Adsorb other IgM antibodies*

Older Terminology Describing Antigen Expression

• 1971, Marsh et al, Described mosaic nature of I antigen

• ID

• Poorly developed on cord cells

• Gradually acquired during first 18 months

• IF

• fetal origin of I antigen, fundamental to all human red cells

• 1965, Curtain et al

• IT

• Unusual antigen expression

• Cord cells >>adult cells >>> iadult cells

Conventional Name

ISBT Symbol

Allele Name

Phenotype

ABO blood group

ABO (001)

ABO*A1 A1

ABO*A2 A2

ABO*A3 A3

ABO*AW Ax & Others

ABO*AEL Ael

ABO*AM Am

ABO*B01 B

ABO*BW Bsubgroups

ABO*O.01 O

ABO*O.02 O

ABO Blood Group System ISBT Terminology

http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/

Gene to Gene Product (Transferase)

Globular Head A

B

mRNA

GalNAc13Gal-R |2 Fuc

Gal13Gal-R |2 Fuc

9q34….

1 2 3 4 5 6 7 5’ UTR 3’ UTR

Exons

RNA

* * * *

354 amino acids Courtesy of G. Denomme

A & B Transferase Amino Acid Structure

C T S Catalytic Domain NH2 COOH

0 50 100 150 200 250 300 350

Amino Acids

(354)

C = Cytoplasmic Tail

T = Transmembrane Domain

S = Stem Region

C

T

S

Catalytic Domain


10

CHO Antigens

Type II Unbranched Chain

i Antigen

R

ß 1-4

β1,3

β1,4

CHAIN TYPES • Type 1 – ABH antigen in secretions

Gal (ß1-3) GlcNAc (ß1 R)

• Type 2 – Most ABH on RBCs Gal (ß1-4) GlcNAc (ß1 R)

• Type 3 (repetitive A) – Glycoprotein in secretions & Glycolipid on RBCs

Gal (ß1-4) GlcNAc (ß1-4) Gal (ß1-4) GlcNAc (1 R)

• Type 4 (globo-A) Gal (ß1-3) GalNAc (1-4) Gal (1-4) Gal (ß1-4) Glc - Cer

-2-L-fucosyltransferase

1-4

Type II

H (FUT1) GENE

H ANTIGEN

Chromosome 19


1-3

Type I

Se (FUT2) GENE

Type 1H ANTIGEN

Chromosome 19


11

CHO Antigens

H4 (Type 4)

- Gal - GlcNAc – (Gal – GlcNAc)n - Gal - Glc - R H4

Gal - GlcNAc

Fuc

Gal - GlcNAc

Fuc Gal - GlcNAc

Fuc

H DEFICIENT PHENOTYPES

PHENOTYPE GENES RBCs SALIVA Oh H-deficient hh,sese (ABO) None None Non Secretor Oh Partially hh, sese (ABO) Trace H None Deficient A or B Non Secretor H-deficient hh, Se, OO Trace H H

Secretor, OO H-deficient hh, Se, A or B Trace H, A or B

Secretor A or B A and/or B

A Antigen

GalNAC

-3-N-acetylgalactosaminyltransferase

A gene

Gal

B Antigen

-3-D-galactosyltransferase

B gene

H Antigen Expression

Most H Least H

O > A2 > B > A2B > A1 > A1B > H Deficient


12

CHO Antigens

ABO Histo-Blood Group Antigens

• Tissues

–Found in all organs of body

• Platelets

• Environment

–Bacteria

–Animals

ABO Antibodies

• Antibody production begins after birth

• “Naturally occurring” or non-red cell stimulated

• Immune response to gut & environmental bacteria (Enterobacteriaceae)

• Antibodies detected at 3-6 months, adult levels at 5-10 years old

• Antibody levels vary between adults – Titers vary from 4 to 2048 or greater

• Depends on stimulation – Multiparous women have higher titer

– Bacteria-based nutritional supplements can increase titer

– Processed foods can result in decreased titer

• An individuals ABO titer may vary over time

Anti-A & Anti-B

• Non-Red Cell Stimulated “Naturally occurring”

– Anti-A titers are higher than anti-B titers.

– Anti-A and -B titers are higher in O persons than in A or B people.

• Immune

– Group A & Bs have some IgG (often IgG2), most IgM

– Group Os have IgG & IgM

– Some IgA

Anti-A & Anti-B

Serologic Characteristics

• Optimum temperature of reactivity

– IgM - 4C

– IgG - 4C & 37C

• Can cause in vitro hemolysis

Clinical Significance

• Efficient activator of complement

• Cause intravascular hemolysis

• Cause HTR

• Mild HDFN (group O moms w/ A or B babies)

Lectins with ABH Specificity

Plant Seeds

• Anti-B

– Bandeiraea simplicifolia

• Anti-A1

– Dolichos biflorus (diluted)

• Anti-H

– Ulex europaeus

• Anti-A

– Dolichos biflorus (undiluted)

– Phaseolus limensis

Other Stuff

• Snails

– Anti-A

• Helix pomatia

• Eel

– Anti-H

• Anguilla japonica

Number of A & B Antigens per RBC

RBC Type Measured Calculated

A1 Adult 810,000 to 1.170,000 > 2,000,000

A1 Cord 250,000 to 370,000

A2 Adult 240,000 to 290,000

A2 Cord 140,000

A1B Adult 460,000 to 850,000 A 310,000 to 560,000 B

B Adult 610,000 to 830,000 > 2,000,000


13

CHO Antigens

Gather

Patient

Info

Forward Type Discrepancy

Missing or Weak Antigen

Subgroup of A/B Suppressed Antigen

Unexpected or Extra Antigen

Acquired B

B(A)

mf

Ab Coated

Extra or Missing antigen?


A1 AND A2 PHENOTYPES

• A2 phenotype mutation of A1 transferase gene

• Quantitative difference

• Number of antigen sites

• Qualitative difference

• A2 can make anti-A1

• Transferase activity differs

• A1 transferase adds GalNAc to Type 4 globo-series chains (glycolipids)

Serologic Characteristics of A Subgroups

Subgroup Anti-A Anti-A,B Anti-A1 Anti-H Secretions

A2 4+ 4+ 0 3+ A & H

Aint 4+ 4+ 2+ 3+ A & H

A3 mf mf 0 3+ A & H

Ax 0/w +/++ 0 4+ H

Am +w +w 0 4+ A & H

Aend +w +w 0 4+ H

Ael 0 0 0 4+ H

Abantu 1mf 1mf 0 4+ H

Afinn +w +w 0 4+ H

Serologic Characteristics of B Subgroups

Subgroup Anti-B Anti-A,B Serum Contain Secretions

B3 +mf +mf No anti-B H

Bx 0/+ 0/++ No anti-B B & H

Bm +/++ +/++ Anti-B B & H

Investigation of Weak A Subgroups

Weakly positive w/ Anti-A

Possible A3, Ax, Aend

Mixed Field <10% mf Weak with Anti-A,B

A3 Aend Ax

Adapted from Harmening, 6th ed 2012


14

CHO Antigens

Investigation of Weak A Subgroups

Negative w/ Anti-A

Possible Am, Ay, Ael

Secretes A & H Secretes

weak A & H Secretes H only

Am Aend Ael

Adapted from Harmening, 6th ed 2012

Adsorbs & Elutes Anti-A or –A,B

Saliva Study Hemagglutination Inhibition

Anti-A + Saliva

Anti-B + Saliva

Anti-H + Saliva

A1 cells 0

B cells 4

O cells 0

Acquired B

Anti-A Anti-B Anti-A1

lectin

A1 cells B cells

4+ 1-3+ 4+ 0 4+

HOH

CH2OH

HO

HO

D-galactose D-galactosamine

HOH

CH2OH

NH2

HO

N-acetyl-D-galactosamine D-galactosamine

Deacetylase

B(A) Extra A Antigen

Anti-A Anti-B A1 cells B cells

W-4+ 4+ 4+ 0

GalNAC

-3-N-acetylgalactosaminyltransferase

“Super “B gene (mutations in B gene)

Extra Antigen

“A-like”

Antibody Coated RBCs Spontaneous Agglutination

After Centrifugation

Anti-A Anti-B Ct A1 cells B cells

1+ 4+ 1+ 4+ 1+


15

CHO Antigens

Reverse Type Discrepancy

Missing or Weak Antibody

Weak Anti-A Weak Anti-B

Unexpected or Extra Antibody

Anti-A1

Autoantibody

Alloantibody

Rouleaux

Passive Antibody

Extra or Missing antibody?


Testing RBCs to Resolve ABO Discrepancies

• Type with:

– Anti-A,B

– Anti-A1 lectin

– Different manufacturer of Anti-A,-B, -A,B

– Acidified anti-B

• Incubate –A, -B, -A,B at RT

– Use with caution since this is not per manufacturer’s directions

• Treat to remove antibody

• Look at RBCs in plasma to assess for rouleaux

• Phenotype patient if alloantibody is identified

Testing Serum/Plasma to Resolve ABO Discrepancies

• Test O cells (Screening Cells) at IS

• Perform “mini” cold panel

– Cord cells

– Autocontrol

– ABO compatible A1, A2, B cells

– Incubate at RT, 18C, 4C

• Prewarm reverse type

• Test antigen negative A1, A2 or B cells corresponding to alloantibody identified

• Saline replacement (rouleaux)

LEWIS ANTIGENS

ANTIGEN ISBT SYMBOL ISBT No.

Lea LE1 007001

Leb LE2 007002

Leab LE3 007003

LebH LE4 007004

ALeb LE5 007005

BLeb LE6 007006

Frquency (%) of Lewis Phenotypes

Phenotype Whites Blacks

Le(a+b-) 22 19.5

Le(a-b+) 72 52

Le(a-b-) 6 28.5

Le(a+b+) Rare Rare

Chromosome Genes Inheritance Le 19 Le (FUT 3) Dominant le Amorph Se 19 Se (FUT 2) Dominant se Amorph

-2-L-fucosyltransferase Type I

Se (FUT2) GENE

Type 1H ANTIGEN

Se gene frequency ~80%, varies by ethnicity

FUT2 & LU - 1st example of autosomal linkage & recombination due to crossing-over in man

FUT2 & LU also linked to C3

1-3


16

CHO Antigens

-2-L-fucosyltransferase Type I

Le (FUT3) GENE

Lea Antigen 1-2


Le (FUT3) Gene

Leb Antigen


Se (FUT2) Gene

1-2

1-2

Be able to look at phenotype & determine secretor status

GENES PHENOTYPE

Le Se Le(a-b+)

Le sese Le(a+b-)

lele Se or sese Le(a-b-)

Gal GlcNAc

(ß 1- 3)

Le(a-b-)

Gal GlcNAc (ß 1- 3)

Fuc

Type 1

sese

H Type 1

Se

Practice – Fill in the Blank

Gene Secretions Red Cell Phenotype

A, H, Se, Le

B, H, sese, lele

H, Lea, Leb

B, hh, sese, Le

AB, Le(a-b-)

Practice – Fill in the Blank Answers

Gene Secretions Red Cell Phenotype

A, H, Se, Le A, H, Lea, Leb A, Le(a-b+)

B, H, sese, lele Type 1 chains B, Le(a-b-)

OO, H, Se, Le H, Lea, Leb O, Le(a-b+)

B, hh, sese, Le Lea O, Le(a+b-)

AB, H, Se or sese, lele

A, B, H, Type 1 or Type1H chains

AB, Le(a-b-)


17

CHO Antigens

Leab (LE3) and Anti-Leab

• Inseparable anti-Lea & anti-Leb

• Originally termed Lex (Lex now known as CD15)

• Negative with Le(a-b-) RBCs

• Positive with 90% of cord cells

• Made by A, B or AB, Le(a-b-), secretors

Anti-Lec & Anti-Led

• Lec & Led – Not produced by Le gene

• Antibodies produced in immunized goat • Rare human antibody • Monoclonal antibodies

Anti-Lea & Anti-Leb

• Found in Le(a-b-) individuals

• IgM, naturally occurring, few IgG

• Bind complement

• Optimum temperature of detection

– RT IAT

• Can cause in vitro hemolysis

• Not significant when not active at 37C

• Rarely causes HTR

• Does not cause HDN

TRANSFUSION 2015;55;2486–2488

Anti-LebH Anti-LebL Anti-ALebL

O, Le(a-b+) 3+ 3+ 0

A2, Le(a-b+) 3+ 3+ 3+

A2B, Le(a-b+) 2+ 3+ 2+

B, Le(a-b+) 1+ 3+ 0

A1, Le(a-b+) w 3+ 3+

A1B, Le(a-b+) 0 3+ 3+

P1Pk, FORS1 & Globoside Systems

P1PK System 003

Globoside System 028

FORS System 031

Globoside Collection 209

P1 P1PK1 003001

P GLOB1 028001

FORS1 031001

LKE GLOB3 209003

Pk P1PK3 003003

PX2 GLOB2 028002

NOR P1PK4 003004

Biosynthesis Pathway The Blood Group Antigen FactsBook 3rd Edition 2012


18

CHO Antigens

Phenotypes and Frequencies Adapted from Modern Blood Banking and Transfusion Practices 6th Edition

Phenotype Antigens Possible Antibodies Caucasian Frequency

African American Frequency

P1 P1, P, Pk, (PX2)

None 79% 94%

P2 P, Pk, (PX2) Anti-P1 21% 6%

p None (PX2) Anti-PP1Pk Rare Rare

P1k P1, P

k Anti-P Very Rare Very Rare

P2k Pk Anti-P, Anti-P1 Very Rare Very Rare

P1/P2 Phenotypes Chromosome

22 1

2a

2 3

P1 allele

P2 allele

ACG

ATG TGA

ORF

Dominant Trait - A4GALT*P1.01 C>T nucleotide change results in fewer coding

transcripts P1: Normal amount of enzyme results in P1 & Pk P2: Not enough enzyme for P1 antigen formation

Adapted from The Blood Group Antigens FactsBook 3rd Edition 2012

P1 Phenotype

Lactosylceramide

Paragloboside(aka Type 2 Chain)

P1 PX2

Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal Glu Cer

GlcNAc

β1-1 β1-4

β1-3

Lactotriasylceramide

Gal Glu Cer β1-1 β1-4

Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal α1-4

Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

α1-4 GlcNAc

α4GalT1

A4GALT

P2 Phenotype

Lactosylceramide


P1 - negative PX2

Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal Glu Cer

GlcNAc

β1-1 β1-4

β1-3



Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

α1-4 GlcNAc

α4GalT1

P

Lactosylceramide

P (Globoside)

Pk


α4GalT1


Gal α1-4


Gal α1-4

GalNAc

β 1-4

GLOB (B3GALNT1)

β3GalNAc

A4GALT Other Antigen Frequencies

Antigens Frequency

FORS1 <0.1%

LKE 98%

NOR 2 known families

PX2 >99.9%


19

CHO Antigens

P

Lactosylceramide

P (Globoside)

Pk


α4GalT1


Gal α1-4


Gal α1-4

GalNAc

β 1-3

GLOB (B3GALNT1)

β3GalNAc

A4GALT


Gal α1-4 Gal

NAc

β 1-3

Gal

α1-4

NOR


Gal α1-4

GalNAc β 1-3

GalNAc

β 1-3

FORS1

P

P (Globoside)


Gal α1-4

GalNAc β 1-3

NOR FORS1


Gal α1-4

GalNAc

β 1-3

Gal β 1-3

NeuAc


Gal α1-4

GalNAc

β 1-3

Gal β 1-3

LKE

Gb5

P1k and P2

k Phenotype

Both very rare in all populations

Recessive Traits

Mutations in B3GALNT1 create an absence of the P antigen Mutation in “right leg” as you look at diagram

All Pk individuals have naturally occurring anti-P in their serum, reacting equally with P1

k and P2k

p (null phenotype)

Lacking P1, P, and Pk

Rare recessive inheritance

High consanguinity rate

Missense and nonsense mutations in the A4GALT gene

Stronger PX2 expression

Lactosylceramide


P1

Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal Glu Cer

GlcNAc

β1-1 β1-4

β1-3



Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal α1-4

Pk

α4GalT1


Gal α1-4


Gal α1-4

GalNAc

P

α4GalT1

Lactosylceramide


Gal Glu Cer

GlcNAc

Gal

β1-1 β1-4

β1-3 α1-4

Gal Glu Cer

GlcNAc

β1-1 β1-4

β1-3



α4GalT1

α4GalT1

p (null phenotype)


20

CHO Antigens

Summary

Gene Enzyme Antigen

A4GALT 4-α-galactosyltransferase (α4GalT1)

P1 , Pk ,NOR

B3GALNT Β-1,3-N-actylgalactosaminlytransferase (β3GalNAcT1)

P & PX2

GBGT1 α-1,3-acetylgalactosaminlytransferase (3αNGalncT1)

FORS1

Phenotype Antigens Possible Antibodies

P1 P1, P, Pk (PX2) None

P2 P, Pk (PX2) Anti-P1

p None (PX2) Anti-PP1Pk

P1k P1, P

k Anti-P

P2k Pk Anti-P, Anti-P1

Summary

Summary

Antibody Class HTR HDFN Spontaneous abortion

Anti-P1 IgM (IgG rare) No to mod/delayed(rare)

No No

Anti-P IgM and IgG Yes No to mild Yes

Anti-PP1Pk IgM and IgG Yes No to mild Yes

Anti-PX2 IgM and IgG Unknown Unknown Unknown

Anti-FORS IgM (IgG rare) Unknown Unknown Unknown

Anti-LKE IgM Yes (rare) No No

Anti-NOR IgM Unknown Unknown No

Questions?

[email protected]


21

CHO Antigens

Rh Blood Group System

Sue Johnson, MSTM, MT(ASCP)SBB Director, Clinical Education BloodCenter of Wisconsin

Milwaukee, WI

Objectives

1. Discuss Rh genes, biochemistry, and antigen development.

2. From a Rh phenotype determine most probable genotype using nomenclature of Wiener, Fisher and Race, and Rosenfield/ISBT

3. Define AABB Standards for RhD typing in donors, transfusion candidates and obstetric patients

4. Explain the causes and clinical significance of serologic weak D antigen. – Weak D types

– Partial D

– Del

5. Discuss compound antigens.

6. Differentiate anti-D, anti-C and anti-G.

Objectives

7. Describe partial e antigen including hrS and hr B and recognize the importance of individuals with variant e antigen.

8. Recognize the importance of low frequency antigens in

identifying genes (gene complexes) with altered D, Cc, and Ee

expression.

9. Describe serologic and hematologic findings, and antibodies

of Rhnull and deletion phenotypes.

10. Discuss the clinical significance of Rh alloantibodies and the unique characteristics of Rh reagents.

11. Discuss the relationship between the Rh and LW blood group systems.

rh’

hr’

Rho

rh”

hr”

Weiner Theory of Rh Inheritance

D

d

C

c

E

e

rh’

Fisher-Race Theory of Rh Inheritance

Rho D

rh’ C

rh” E

hr’ c

hr” e

Weiner to Fisher-Race Nomenclature

R1 DCe

r ce

R2 DcE

Ro Dce

r’ Ce

r” cE

Rz DCE

ry CE

Modified Weiner Haplotype

Fisher-Race Haplotype

Fisher-Race Haplotypes of the Rh System

Prevalence (%) Haplotype White Black Asian

DCe 42 17 70

dce 37 26 3

DcE 14 11 21 Dce 4 44 3 dCe 2 2 2

dcE 1 <0.01 <0.01

DCE <0.01 <0.01 1

dCE* <0.01 <0.01 <0.01

AABB Technical Manual. p. 320


22

Rh

Prevalence of Rh Genotypes

Whites Blacks

Genotype Prevalence (%) Prevalence (%)

DCe/dce (R1r) 33

DCe/DCe (R1R1) 18

dce/dce (rr) 15

DCe/DcE (R1R2) 12

DcE/dce (R2r) 11

DcE/DcE (R2R2) 2

Dce/dce (Ror) 22.9

Dce/Dce (RoRo) 19.4

Rh Haplotype to Rh Genotype

8 haplotypes 36 genotypes

R1R1 R1r R1ry RoRz r’ry

R1r’ R1Ro R2Rz Rory r”ry

R2R2 Ror’ Rzr” r’r’ r”r’

R2r” R2r R2ry r”r”

Ror R2Ro R1R2 rr

RoRo Ror” R1r” ryry

RzRz R1Rz R2r’ r’r

Rzry Rzr’ Rzr r”r

Weiner Haplotype Terminology

Symbol D C E c* e* Shorthand Designation

R 1 + + 0 0 + R1

r ' 0 + 0 0 + r'

R 2 + 0 + + 0 R2

r '' 0 0 + + 0 r''

R Z + + + 0 0 Rz

r y 0 + + 0 0 ry

R O + 0 0 + + Ro

r None 0 0 0 + + r

Interpretation of Rh Typing

Possible Most Probable -D -C -E -c -e Phenotype Genotypes Genotype

+ + + + + D, C, c, e DCe/dcE (R1r”)

DCe/DcE (R1R2)

Dce/dCE (Rory)

DCE/dce (Rzr)

DCE/Dce (RzRo)

DcE/dCe (R2r’)

R1R2

Rosenfield Nomenclature

• System based on serologic observations

• Antigens numbered in order of discovery or assignment to the Rh system

• Presence of antigen is indicated by appropriate number following Rh: (Ex. - Rh:1 is D+)

• Absence of antigen is indicated by a negative sign preceding a number (Ex. - Rh:-1 is D-)

Practice Worksheet

Phenotype Fisher-Race Weiner Rosenfield/ISBT

D+C+E-c+e+

D+C+E-c-e+

DCe/DcE

R2r

Rh:1, -2,-3, 4, 5

D-C-E-c+e+


23

Rh

Practice Worksheet - Answers

Phenotype Fisher-Race Weiner Rosenfield/ISBT

D+C+E-c+e+ DCe/dce R1r Rh:1, 2, -3, 4, 5

D+C+E-c-e+ DCe/DCe R1R1 Rh:1, 2, -3, -4, 5

D+C+E+c+e+ DCe/DcE R1R2 Rh:1, 2, 3, 4, 5

D+C-E+c+e+ DcE/dce R2r Rh:1, -2, 3, 4, 5

D+C-E-c+e+ Dce/Dce or Dce/dce

RoRo or Ror

Rh:1, -2,-3, 4, 5

D-C-E-c+e+ Dce/dce rr Rh:-1, -2, -3, 4, 5

Rh Associated Genes

System No. System Name Gene Name Chromosome

004 Rh RHD 1 Polymorphic

RHCE 1 Polymorphic

030 Rh Associated Glycoprotein

RHAG 6 Monomorphic

RHAG is ancestral gene

2 3 4 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 10

RH Genes – Rh Positive

RHD

RHCE

Chromosome 1

Locus 1 - presence of RHD codes for the presence of D or no D.

Locus 2 - presence of RHCE codes for Ce, CE, cE, ce.

ce RHCE*01 or RHCE*ce Ce RHCE*02 or RHCE*Ce cE RHCE*03 or RHCE*cE CE RHCE*04 or RHCE*CE

D RHD*01.01

11 72

32 53 94 107 158 167 230 231 282 290 347 358

75 131 135 186 201 263 266 321 324 391

417

RhD differs from RhCE by 34 to 37 amino acids = E= C=

RhD vs. RhCE Protein

Adapted from Flegel, Curr Opin in Hemat 2006, 13:476–483

Proline (E) 226 Alanine (e) Serine (C) 103 Proline (c)

RhD Negative

• Deletion of RHD

• Inactivating mutations of RHD

– RHD in African Americans

• Hybrid RHD-CE-D in African backgrounds

9 1 2 3 4 5 6 7 8 10

9 1 2 3 4 5 6 7 8 10

9 1 2 3 4 5 6 7 8 10

Gene Conversion

• Portions of RHCE into RHD

1 2

3 4

6 5

7 9

8 10

5’

3’ 10

9 8

7 5

6 4

2 3

1 5’ 3’

6 5

4 7


24

Rh

Gene Conversion

• Portions of RHD into RHCE

1 2

3 4

6 5

7 9

8 10

5’

3’ 10

9 8

7 5

6 4

2 3

1 5’ 3’

6 5

4 7

4

Missense, Nonsense, Frameshift & Splice Site Mutations

1 2 3 4 6 5 7 9 8 10

5’ 3’

10 9 8 7 5 6 4 2 3 1

5’ 3’

G>C

C>G

RHD

RHCE

Missense – amino acid change Nonsense or Frameshift – prevent expression Splice site – no or reduced expression

C/c E/e

Proteins Required for Rh Expression

RhAg

RhD RhCE

Rh Glycoprotein (RhAG)

• 45-100 kDa

• Carry ABO structures

• Chromosome 6 - RhAG gene

–409 a.a.

• Crossed RBC membrane 12 times

• Absent on Rhnull, U- red cells

RhAG as a blood group

Tilley et. al, Vox Sanguinis (2010) 98, 151–159

Rh Complex

D+, C+, E+, c+, e+

RhD

RhCE LW

CD47

Band 3

GYPA/B

RhAG protein is required for expression of the Rh proteins

RhAG

• RhD, RhCE proteins • RhAG glycoprotein • Band 3 (RBC anion exchanger) is core • LW glycoprotein (ICAM-4) • CD47 integrin-associated protein • Glycophorin A & B • Attached through ankyrin & protein 4.2


25

Rh

http://opm.phar.umich.edu/images/proteins/3b9w.gif

Immunogenicity of D

• Most D-negative individuals lack the entire RhD protein

• RhD and RhCE proteins differ by 32 to 37 amino acids

• Large number of amino acid changes seen as “foreign”

• Explains potent immune response

50-80% of RhD- healthy individuals given >1 unit of D+ blood make anti-D

21-22% of D- patients given >1 unit of D+ blood make anti-D

Weak Expression of D Not at Risk of Making Anti-D

• C in trans with RHD (Ceppellini effect) – r’ haplotype (R1r’ – DCe/Ce)

• Weak D “Types”: amino acid change(s), usually a single change

–Types 1, 2, 3

Ceppelini Effect Not At Risk of Making Anti-D

DCe/Ce ce/ce

Ce/ce DCe/ce DCe/Ce Ce/ce

Ce/ce DCe/ce

DCe/ce ce/ce

Du

Du + +

+

C in trans to RHD

11 72

32 53 94 107 158 167 230 231 282 290 347 358

75 131 135 186 201 263 266 321 324 391

417

Type 2 Gly(385)Ala

Type 1 Val(270)Gly

Account for 80-90% of Weak D Not at risk of making Anti-D

Type 3 Ser(3)Cys

Adapted from Flegel, Curr Opin in Hemat 2006, 13:476–483

Partial D

• Lack exofacial epitopes or have altered exofacial epitopes

–Hybrid proteins

–Missense mutations affecting exofacial protein

Partial D – European Ancestry

• DNB, DVI and DVII most common in European ancestry

• DVI

– 2 reports of fatal hydrops fetalis • Transfusion.1983 Mar-Apr;23(2):91-4

• Obstet Gynecol. 2003 Nov;102(5 Pt 2):1143-5 2003

– Anti-D reagents designed to be neg at IS/pos at IAT

IS D IAT Ct. IAT

Anti-D 0 3 0


26

Rh

Partial D – African American

• DIIIa & DIVa most common – Type as RhD positive at IS

IS

Anti-D 3+

Partial D Hybrid Alleles Associated Low Prevalence Antigen

Partial D Variant Low Prevalence Antigen

DIIIa DAK

DIVa Goa

DVa Dw

DVI-2, -3, -4 BARC

DVII Tar

DFR FPTT

DBT Rh32

Partial D Categorization

• Serologic

– A,B,C,D

– II to VII, DFR, DBT

• Molecular

– Types 1…

– DAU - cluster

Monoclonal Anti-D Panel

Interpretation: DVI

Bagene Weak D Worksheet

Deletion of exon 9 in Asians occurs in 10-30% European Ancestry – 0.027%

Del

•Type as D-negative (IS & IAT), only adsorb & elute anti-D •Severely reduced protein •2 individuals have made anti-D after receiving D+ blood


27

Rh

D Epitope on RHCE Genes

• Crawford (ceCF) phenotype

• ceHAR – formerly known as R0Har, DHAR

1 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 10

Locus 1 Locus 2

Exons

No D antigens ce antigens

RHCE

ceCF results from 3 nucleotide changes, 48G>C, 697C>G, 733C>G in RHce gene.

D Epitope on RHce Gene - ceCF

IS

Anti-D 3+

Anti-D Reagents: Reactions with Crawford Phenotype RBCs

Anti-D RBCs

Reagent IgM IgG Crawford

GammaClone GAMA401 F8D8 Pos Immucor-4 MS201 MS26 Neg Immucor-5 TH28 MS26 Neg Ortho Bioclone MAD2 Human

polyclonal Neg

Ortho (ID-MTS) MS201 Neg

1 3 4 5 6 7 8 2 10 9 8 7 6 5 4 3 1

Locus 1 Locus 2

Exons

No D antigens ce antigens

RHCE

ceHAR results from one RHD exon inserted into the RHCE gene.

D Epitope on RHCE Gene - ceHAR

IS

Anti-D 3+

ceHAR Phenotype: Reactivity with Reagent Anti-D

Anti-D RBCs

Reagent IgM IgG ceHAR

Gamma-Clone GAMA401 F8D8 Pos*

Immucor-4 MS201 MS26 Pos* Immucor-5 TH28 MS26 Pos*

Ortho Bioclone MAD2 Human polyclonal

Neg

Ortho (ID-MTS) MS201 Pos Biotest (Bio-Rad) BS232 BS221

H41 11B7 Pos

Quotient - Alpha LDM1 Pos Quotient - Delta LDM1 ESD1M Pos

*Positive reactions often weaker at IAT

Do you want to detect Weak/Partial D?

• Blood Donors Yes

• Pretransfusion Testing No

• Prenatal Patients ??

• Infants of Rh-Negative Mother Yes


28

Rh

Reasons to Resolve Weak D Expression

Pregnancy • Avoid giving RhIG to women who do not need it

(Rh status is confirmed for historical discrepancies)

• Resolve early in pregnancy to eliminate false-positive rosette tests

Negative Control Positive Control Weak D+ Mom

Low Resolution

High Resolution

Serologic Weak D Phenotype Detected

Molecular Testing Appropriate for

Phenotype

Molecular Testing includes weak D type and partial D assays

Molecular Testing explains phenotype

Yes No

Sequencing

Result Reported

Adapted from Wagner, F. Perspectives in Transfusion Medicine, Issue 5, Grifols, 2015.

Immune Response to RhD

• 1st exposure slow, up to 4 weeks

–Short primary IgM response

• Memory lasts for years after immunizing event

• Response on re-stimulation

–Strong IgG, often within 24 hrs

–Peaks quickly (~ 6 days)

Decreasing Order of D Antigen Strength

~D Antigens/RBC*

D-deletion Most D--, Dc-, DCw- DcE/DcE (R2R2) DCe/DcE (R1R2) DCe/DCe (R1R1) 14,500–22,800* DcE/dce (R2r) 12,000-19,700* DCe/dce (R1r) 9900-14,600*

*Wagner, et al. Blood 2000;95:2699-2708

Characteristics of Rh Antibodies

• Immune - IgG • Most are IgG1 or IgG3

• Detectable antibody persists

• Some Rh antibodies occur together

• Rare non-RBC stimulated - IgM

Anti-E Anti-CW Anti-CX

• IgM - R.T. and 37ºC • IgG - some 37ºC, most

IAT • Enhanced by:

• High protein media • Proteolytic enzymes • Gel test • Polybrene • PEG

• Antibodies seldom show dosage

• Rarely bind C in vitro

Anti-D Reagents

• High Protein

– Slide and modified tube

• IgG anti-D

• 20-24% albumin +

additives

• Rh control required

• Low Protein*

– Saline reactive tube test • Polyclonal IgM anti-D

– Chemically modified

• Polyclonal IgG anti-D • Interchain disulfide

bonds reduced

– Monoclonal Blend • Monoclonal IgM anti-D

+ polyclonal IgG anti-D

– Monoclonal • Monoclonal IgM anti-D

*6-8% Albumin - No Rh Control


29

Rh

RH gene diversity • Gene conversion:

– Portions of RHCE into RHD

– Portions of RHD into RHCE

• RHD: > 200 alleles

– Normal D RHD*01.01

– RHD*01.68 & counting

• RHCE: > 80 alleles

– ce RHCE*01 or RHCE*ce

– Ce RHCE*02 or RHCE*Ce

– cE RHCE*03 or RHCE*cE

– CE RHCE*04 or RHCE*CE

1 2

3 4

6 5

7 9

8 10

5’

3’ 10

9 8

7 5

6 4

2 3

1 5’ 3’

6 5

4 7

4

“Compound” Antigens & Corresponding Antibodies

f

rhi

cE

CE

ce Ce Rh27 Rh22

Antigens expressed on a single Rh protein

Anti-f – remember how to identify & what Rh phenotypes are appropriate for transfusion Recognize how you can use these antibodies to determine haplotype – D+C+E-c+e+f+, rhi+ DCe/dce

Cw & Cx • Most Cw+ and Cx+ are C+

– R1-like DCCwe or DCCxe

• R0-like DCwce

• Frequencies: Cw+ 1%

Cx+ <1%

• Cw – 122 A>G

• Cx – 106 G>A

• Mar is antithetical to both, high prevalence antigen

• Anti-MAR is non-reactive with:

Rhnull D--

Cw Cw

Cx Cx

Cw Cx

G and Anti-G

• G Antigen

– Exon 2 103Ser

• RHD gene

• RHCe gene

• RHCE

• Most C+ or D+ RBCs are G+ (Rh12)

• Anti-G appears to be anti-D and anti-C

• Explains Anti-D and anti-C in D- patients who have

received D- RBCs

Differential Adsorption/Elution Anti-D, Anti-C or Anti-G?

Anti-G Negative Second Eluate

RO (D+ G+)

Second Adsorption

Anti-G, -C Anti-C First Eluate

First Adsorption r' (C+ G+)

ANTI-G, -C ANTI-D, -C

Adsorptions and Elutions

Patient Sample

1st Adsorption (r’)

Adsorbed Serum (1st) Eluate

2nd Adsorption (Ro)

Adsorbed Eluate (2nd) Eluate


30

Rh

Patient with Anti-D & Anti-G

G+

r‘ (D-C+G+)

C+

C+

G+

G+

r‘ (D-C+G+)

C+

C+

G+

Anti-D

Anti-G

r‘ (D-C+G+)

C+

C+

Anti-D

Anti-G:G+

Anti-G:G+

r‘ (D-C+G+) Adsorbed

Serum

Anti-D

r‘ (D-C+G+)

Eluate

Anti-G

Anti-G Anti-D

Anti-G

Courtesy of MG Knier

Patient with Anti-D & Anti-G

G+

Ro (D+C-G+)

D+

D+

G+

G+

Ro (D+C-G+)

D+

D+

G+

Anti-G

Anti-G

Ro (D+C-G+)

D+

D+

Anti-G:G+

Anti-G:G+

Ro (D+C-G+) Adsorbed

Eluate

Anti-G

Ro (D+C-G+)

Eluate

Anti-G


Patient with Anti-D & Anti-G Summary

r' (D-C+G+) Adsorbed

Serum

r' (D-C+G+)

Eluate

Anti-G

Anti-G

Ro (D+C-G+) Adsorbed

Eluate

Anti-G

Ro (D+C-G+)

Eluate

Anti-G

Anti-D

Anti-D Anti-G


“Partial e” with Anti-e

• Most often found in African Americans

• e+ and produce e-like antibodies

• Initial immune response anti-e-like

– Anti-f (-ce) Anti-hrS

– Anti-Rhi (-Ce) Anti-hrB

Rh MNS P Lewis Kell Duffy Kidd

D C E c e f M N S s P1 Lea Leb K k Fya Fyb Jka Jkb IAT

1 + + 0 0 + 0 + + + + 0 + 0 0 + 0 + 0 + 0√

2 0 0 0 + + + + 0 + 0 + 0 + + 0 + + + 0 2+

3 0 0 + + 0 0 0 + 0 + + 0 + + + + 0 0 + 0√

4 + + 0 0 + 0 + 0 + + 0 0 0 0 + + + + + 0√

5 0 0 + + + + + + 0 + 0 0 + 0 + 0 + 0 + 2+

6 0 + 0 0 + 0 + + 0 + + 0 + + + + + + + 0√

7 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + + 0 0√

8 0 0 0 + + + + 0 + 0 + 0 + 0 + + 0 0 + 2+

9 + + 0 + + + + + 0 + + + 0 0 + 0 + + 0 2+

10 + 0 + + 0 0 0 + + + + + 0 0 + + 0 + 0 0√

11 + 0 0 + + + 0 + 0 0 + 0 + 0 + 0 0 + + 2+

A 0√

Rh Phenotype: D+ C- E- c+ e+


PEG /

Gel /

Enz


1 + + 0 0 + 0 + + + + 0 + 0 0 + 0 + 0 + 3+

2 0 0 0 + + + + 0 + 0 + 0 + + 0 + + + 0 3+

3 0 0 + + 0 0 0 + 0 + + 0 + + + + 0 0 + 2+

4 + + 0 0 + 0 + 0 + + 0 0 0 0 + + + + + 3+

5 0 0 + + + + + + 0 + 0 0 + 0 + 0 + 0 + 3+

6 0 + 0 0 + 0 + + 0 + + 0 + + + + + + + 2+

7 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + + 0 2+

8 0 0 0 + + + + 0 + 0 + 0 + 0 + + 0 0 + 3+

9 + + 0 + + + + + 0 + + + 0 0 + 0 + + 0 3+

10 + 0 + + 0 0 0 + + + + + 0 0 + + 0 + 0 2+

11 + 0 0 + + + 0 + 0 0 + 0 + 0 + 0 0 + + 3+

A 0√

Rh Phenotype: D+ C- E- c+ e+


31

Rh

“Partial e” Anti-e-like Specificity

• -hrS (anti-f-like)

– Broaden or separate anti-Hr (-RH18)

• May include anti-D

• -hrB (anti-Ce-like)

– Separate anti-HrB (-RH34) – 2009

• May include anti-D

• Other variants

• Antibodies are not all the same

D(C)ceS (r’S or ceS)

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

RHD RHCE

D-

positive

1 2 3 4 5 6 7 8 9 10

ceS

(hrB -)

VS+, V- W16C L245V G336C

Variant RHce

VS+V-hrB-HrB-

Hybrid RHDIIIa-RHCE-RHDIIIa

Variant C, D-

RN

• Ce – D – Ce protein

• Weak C and e, (C)(e)

• Enhanced D

• Rh32+

RHD RHCE

D-Deletion Types

Partial Deletion Can Make: D-- Anti-Rh17 D•• Low: Evans Anti-Rh17 Dc- DC- Anti-Rh17

AMORPH – Rhnull

D-C-E-c-e-

r r

D+C-E+c+e-

Apparent Genotype R1R1

Real Genotype R1 r

D+C-E+c+e-

R2R2

R2 r

D+C-E+c+e-

Apparent Genotype R2R2

Real Genotype R2 r

r = Common RHD deletion & Mutant RHCE

Regulator – Rhnull

R1r

Mutant RHAG

R1R1

Mutant RHAG

Rhnull

R1r or R1R1

Mutant RHAG

rr

RHAG

R1r

Mutant RHAG

Reduced Rh antigens

Normal Rh antigens

No Rh antigens


32

Rh

Rhnull and Rhmod

Phenotype Rh Protein RhAG Altered Gene

Rhnull

Amorph Absent Reduced

(20%) RHCE RHD deleted

Rhnull

Regulator Absent Absent RHAG mutation

RHAG deleted

Rhmod Reduced Reduced RHAG mutation

• No LW or Fy5, reduced GPB, reduced CD47 • Rhnull make anti-Rh29 • Rhnull individuals have compensated

hemolytic anemia - stomatocytes

Normal Rh Antigen Expression Review

Rh Protein

C

E

c

e

f

Ce

cE

CE

G

hrB

hrS

HrB (Rh34)

HrO (Rh17)

Hr (Rh18)

Rh29

RhCe + 0 0 + 0 + 0 0 + + + + + + +

Rhce 0 0 + + + 0 0 0 0 + + + + + +

RhcE 0 + + 0 0 0 + 0 0 0 0 + + + +

RhCE + + 0 0 0 0 0 + + 0 0 + + + +

LW Blood Group System (016)

• 3 antigens

– 005 LWa

– 006 LWb

– 007 Lwab

• All Rhnull are LW(a-b-)

• LWa and LWb antigens are weaker on D- RBCs than D+ RBCs

• All cord cells (D- and D+) are strongly reactive

• Transient LW antigen depression can occur

Anti-LW

• Shows relative anti-D specificity

• No difference in reactivity with D+ or D- cord

cells

• D-, LW+ units may be transfused in most cases

• Non-reactive with AET/DTT treated RBCs


PEG /

Gel /

Enz


1 + + 0 0 + 0 + + + + 0 + 0 0 + 0 + 0 + 3+

2 0 0 0 + + + + 0 + 0 + 0 + + 0 + + + 0 0√

3 0 0 + + 0 0 0 + 0 + + 0 + + + + 0 0 + 0√

4 + + 0 0 + 0 + 0 + + 0 0 0 0 + + + + + 3+

5 0 0 + + + + + + 0 + 0 0 + 0 + 0 + 0 + 0√

6 0 + 0 0 + 0 + + 0 + + 0 + + + + + + + 0√

7 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + + 0 0√

8 0 0 0 + + + + 0 + 0 + 0 + 0 + + 0 0 + 0√

9 + + 0 + + + + + 0 + + + 0 0 + 0 + + 0 3+

10 + 0 + + 0 0 0 + + + + + 0 0 + + 0 + 0 3+

11 + 0 0 + + + 0 + 0 0 + 0 + 0 + 0 0 + + 3+

A 2+

Rh Phenotype: D+ C- E- c+ e+ Possibilities • Partial D with Anti-D • Anti-LW • Autoanti-D

Questions?

[email protected]


33

Rh